Microtubules (MT) are tubular protein structures found in all eucaryotic cells. They are involved in cell motility, cell transport, mitosis, and other cell functions. MT are composed primarily of two related protein subunits termed alpha and beta tubulin (Tu). The MT protomer is a heterologous dimer consisting of an alpha and beta Tu. In vivo, MT exist in a dynamic equilibrium with the protomer. A number of agents have been found to alter MT structure, both in vivo and in vitro. These include low temperature, nucleotides, divalent cations, and plant alkaloids. Many of these agents bind to Tu and alter its assembly properties. Nucleotides play a critical role in Tu assembly, however the mechanism whereby they do so is nucelar. Using fluorescent nucleotide analogs which we have developed we will determine whether the single tightly bound divalent cation present in Tu is a part of or near the exchangeable nucleotide binding site. Using these analogs, and others which we will synthesize, we will examine the kinetics and mechanism of nucleotide binding using fluorescence stopped-flow. The effects of nucleotide binding on Tu conformation will be further explored using proteolysis, chemical crosslinking, and resonance energy transfer. Estimates of the distance between various ligand binding sites will be determined by resonance energy transfer. The thiophosphate nucleotide analogs, GTP-gamma-S and the diastereomers of GTP-beta-S, will be used to attempt to determine the correlation, if any, between nucleotide hydrolysis and Tu assembly. The nucleoside diphosphokinase present in MT preparations will be purified and compared with highly purified Tu with respect to their ability to use the diastereomers of GTP-beta-S as substrate and for assembly. This may enable us to determine whether the GTPase activity present in Tu preparations is an intrinsic activity of Tu.